This project will elucidate critical processes that precisely edit mt mRNAs in Trypanosoma brucei and its regulation and differential editing between life cycle stages. It examines the editosome endonuclease subcomplex which discriminates among thousands of editing sites (ESs), initiates editing and is a likely point of regulation. It uses novel cell lines and methods to examine three editosome endonucleases and their associated proteins and the roles of their domains, interactions and posttranslational protein modifications. It characterizes: 1. Editosome endonuclease ES specificity in vivo by determining the effects on editosome integrity, composition and function and on edited mRNAs of eliminating each endonuclease and mutating their RNase III, Zn finger and PUF domains in bloodstream (BF) and procyclic (PF) stages and in a BF line that survives without editing. It will quantify all editing in BFs and PFs, in vivo endonuclease specificity and if it differs between lif cycle stages. 2. The roles of endonuclease associated proteins by approaches similar to Aim 1. These include B4 and B5 and their RNase III, PUF, Zn finger domains to determine if their effect on endonuclease activity and mRNA cleavage; B6, B7 and B8 and their Zn fingers to determine their effects on endonuclease specificity and regulation; and the low abundance B9 and B10 that transiently interact with editosomes and may regulate their functions. These studies will determine the roles of these proteins in the specificity and regulation of ES selection and cleavage and between life cycle stages. 3. Physical and function interactions between endonucleases and associated proteins by complimentary in vivo and in vitro protein crosslinking methods. It will examine the roles of protein interactions in ES selection, steps of editing, editing of multiple sites and between life cycles. It will characterize the physical and functional organization of editosomes whether proteins exchange between editosomes during editing especially when editing of insertion and deletion ES specified by one gRNA. 4. The roles of editosome protein post-translational modification (PTM) by identifying sites of phosphorylation, methylation, ubiquitination and sumoylation. It will assess roles of PTM in editing and its regulation by examining the effects of mutating selected PTM sites on editosomes and editing and between life cycle stages. These studies will determine whether PTMs regulates steps in editing and editing between life cycle stages. Overall, the project will expand the understanding of a process that is essential for the parasite and may provide drug targets.